Novel gold nanoparticle clusters conjugated with DNA (DNA-AuNP Clusters), which were synthesized by controlling the cross-linking capability of dithiothreitol (DTT) and particle-stabilizing effect of monothiol DNA, were applied for the immobilization and direct electrochemistry studies of Mb. The DNA-AuNP Clusters-based biocomposite film modified electrode was fabricated through layer-by-layer coating. The morphology, structure and electrochemical properties of the DNA-AuNP Clusters and their biocomposite film were characterized by dynamic light scattering, field emission transmission electron microscopy, UV-vis spectroscopy, electrochemical impedance spectroscopy and cyclic voltammetry. The Mb immobilized in the composite film kept its bioactivity and exhibited greatly enhanced direct electrochemical behavior, which is mainly attributed to the good conductivity and excellent biocompatibility of DNA-AuNP Clusters. The modified electrode displayed good electrocatalytic activity to the reduction of H2O2. Therefore, with combining the advantages of the AuNPs and DNA, this DNA-AuNP Clusters-based composite film could be used as a new promising platform for protein immobilization and novel biosensor fabrication. (C) 2012 Elsevier B.V. All rights reserved.